The present invention relates to nucleic acids comprising sequences encoding Ehrlichia ruminantium epitopes that induce a CD4 immune response, and sequences encoding Ehrlichia ruminantium epitopes that induce a CD8 immune response. The invention also provides for multi-epitope DNA vaccines comprising the nucleic acids and for polypeptides encoded by the nucleic acids of the invention. The invention also relates to uses and methods of eliciting an immune response against heartwater disease in a subject using the nucleic acids, multi-epitope DNA vaccines and polypeptides described.
Heartwater is a tick borne disease of both wild and domestic ruminants caused by the obligate intracellular organism, Ehrlichia ruminantium. It is transmitted by ticks of the Amblyomma species and occurs mainly in Sub-Saharan Africa and the Caribbean Islands. Currently there is no safe and effective vaccine. The only commercial vaccine currently in use is the infection and treatment vaccination using live virulent organisms. This method has a number of practical disadvantages and it does not protect against all the field isolates. Thus, there is an urgent need for alternative vaccines. Research on alternative vaccines like inactivated, attenuated and DNA vaccines has been ongoing. With the exception of the attenuated vaccine these have all had limited success in the field.
DNA vaccines consisting of pathogen-derived immunogens could offer a safe and effective alternative to the current vaccine. Work done previously by the Applicant showed that a DNA vaccine encoding four E. ruminantium open reading frames could provide 100% protection in sheep following laboratory challenge but failed under field conditions. In the preceding studies, reverse vaccinology was applied in order to identify additional vaccine candidates. Five low molecular weight proteins that could induce antigen specific recall cellular immune responses in immune sheep peripheral blood mononuclear cells (PBMC) were identified. However, when tested as a DNA vaccine only partial protection against laboratory challenge was obtained.
Additional vaccine candidates were identified that could induce upregulation of cytokines associated with innate immunity and adaptive cellular immune responses. It is well documented that a cellular T helper 1 (Th1) immune response is crucial in the protection against heartwater. These responses are mediated by CD8+ and CD4+T lymphocytes through the production of the Th1 cytokine, IFN-γ. A successful heartwater DNA vaccine might result from immunogens that can elicit similar immune responses. T cell epitopes, the minimal antigenic units of the whole pathogen protein, are presented by major histocompatibility complex (MHC) molecules that are recognised by the host T lymphocytes. Although these small sequences can induce protective immune responses, some can inhibit such responses or induce immunopathology. Selecting only T cell epitopes that induce protective immune responses for incorporation into a multi-epitope DNA vaccine could result in an effective vaccine.
The Applicant has identified 17 CD4+ T cell and CD8+ cytotoxic T lymphocyte (CTL) epitopes from the following E. ruminantium antigens: Erum0660; Erum5420; Erum1150; Erum7360; Erum7140; Erum7350; Erum7620; Erum8010; Erum7320 and Erum2540. Epitopes were identified in vitro using immune sheep PBMC wherein, five of these epitopes induced positive CTL responses, proliferation of CD8+ T cells as well as production of IFN-γ by these cells and expression of cytokines like IL-18 and TNF-α. Twelve of these epitopes were shown to specifically induce IFN-γ production by memory CD4+ and CD8+ T cells in addition to expression of cytokines like IL-12, TGF, iNOS, IL-2, IL-1a, TNF-α and GM-CSF.
These epitopes were used to construct different multi-epitope DNA vaccines. Multi-epitope DNA vaccines have been constructed and showed effective efficiencies against several pathogens like M. tuberculosis, Eimeria tenelle, Toxoplasma gondii. 
The innate immune system recognises specific molecular structures present in the pathogen in order to activate the adaptive immunity. As subunit vaccines, DNA vaccines lack these molecular structures found in live organisms or attenuated vaccines. Hence, they are often unable to stimulate pathogen-specific adaptive immune responses and have to rely on the incorporation of effective adjuvants to enhance their immunogenicity.
Adjuvants, such as Monophosphoryl lipid A (MPL), can function either as immunostimulants which activate innate immune pathways and aid in the enhancement of adaptive immune responses or as vehicles for antigen delivery (e.g. microparticles) which improve delivery of vaccines to the immune system. MPL adjuvant, a derivative of lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) which is one of the innate receptors resulting in activation of multiple innate functions that will support activation of adaptive immune responses. Microparticles can be used to adsorb or encapsulate DNA vaccines in biodegradable particles such as Poly Lactic-co-Glycolic Acid (PLGA). PLGA microparticles have been reported to improve delivery of DNA to antigen presenting cells (APC), enhance gene expression and to protect the DNA against nuclease degradation. Additionally, PLGA microparticles can be formulated for sustained release of DNA over a prolonged period often resulting in improved duration of vaccine induced immunity.